InterLab Human Practices Collaborations

Introduction

The use of horseshoe crabs in the biomedical industry is a widespread problem that affects many different aspects of the world. We wanted to reach out to experts in the field for suggestions on our project and how to mitigate this problem. We also wanted to reach out our community to educate people about the issues that come with horseshoe crab harvesting and synthetic biology as a whole.

Research

Professional Input

Tiffany Black from the Florida Fish and Wildlife Conservation Commission provided some insightful feedback on our project. In her email, she highlighted how education is the key to protecting wildlife. This enables people to appreciate the wildlife around them and to be more likely to protect their environment. However, she described that this is not as easy as it sounds, since people “want to have their cake and eat it too”. A viable alternative to LAL testing may be beneficial to horseshoe crab populations but in consequence may be very expensive. The same applies for the current LAL testing practices. The horseshoe crab blood harvest is currently the most efficient way to achieve endotoxin detection through LAL.

Finding a balance between these two aspects of LAL testing, cost and environmental consequence, is extremely important. If the decline in the horseshoe crab population is so great that their blood can no longer sustain the biomedical industry, then the industry will have to find a more expensive alternative with the consequence of decimating the horseshoe crab population as well.

Black also mentioned the controversies of LAL testing. She stated that biomedical companies do their best to minimize the mortality rates of horseshoe crabs during harvest but the average mortality rate in North America is still about 15%. She also touches on the animal testing to determine the effectiveness of drugs on humans. There have been ethical issues about this practice, but as long as the only alternative is human testing, it is a necessary practice. Black then discusses the illegal capture of horseshoe crabs. She voices the struggles of getting a species protected; in order to achieve protected status, data must be gathered and stakeholders must show interest.

As Tiffany Black, of the Florida Fish and Wildlife Conservation Commission, explained to our team on United States’ regulations and species protection:

“Unfortunately it takes quite a while to get something protected. There needs to be scientific data gathered to determine the declines (and that research takes money and time) and then agencies and governments decide to do something about it, generally from lobbying from “stakeholders” or groups interested in a species’ protection. Then regulations occur based on the data. This would include creation of permits, quotas, and other limiting factors in harvest, and then enforcement of those. South Carolina, and New Jersey, for instance have banned horseshoe crab harvest from their states, even though it is legal elsewhere. So I think all three [biomedical companies, governments and people] should all take part in it.”

Black ends by bringing it all back to the importance of education. She relays the story of a woman from the Delaware Bay area who loves horseshoe crabs. This woman saw a presentation on horseshoe crabs and was told that they could be saved by flipping them over if they were stranded. She went on to save 25,000 as of 2009 and saving more to date. If the environmental education of one woman made such a big difference, then the impact of widespread education could be enormous.

“So, in closing to you, my friends in this ‘environmentally conscious organization’ …in so many cases, things have to start in your homes, in your heads and in your hearts. You have the power to try to become engaged in your own impacts on the planet. We used to say ‘recycle, reuse, reduce.’ My personal favorite is ‘reduce, reduce reduce.’ Reduce your plastic. Reduce your fossil fuel use. Reduce your water use. Read labels, and LEARN about your wild places and how to fit into them with a lessened impact. In just trying, you are doing so much. In wanting to even entertain the alternative LAL idea, you could go on to do great things.”

We also got responses and feedback for our project from various other experts:

Dr. Thomas Novitsky, the CEO of Cape Cod Biosystems, Inc. and Dr. Stanley Watson who learned how to make LAL from its discoverer, Dr. Jack Levin. Dr. Novitsky gave us the perspective of those who rely on the current practice of bleeding horseshoe crabs. While this goes in opposition to our project, it gave us some meaningful feedback about how our project could be improved and what we need to consider in future steps. Dr. Novitsky was able to point out the possible shortcomings of our project and gave us suggestions on how to surpass these issues.

Mark Botton and Paul Shin, the co-chairs of the IUCN (International Union for Conservation of Nature) SSC Horseshoe Crab Specialist Group. In their email they described their goals as a horseshoe crab protection group: to promote the conservation of three Asian horseshoe crab species. They also referred us to useful resources such as horseshoe crab.org, and various books about horseshoe crabs (ie. “The American Horseshoe Crab”, and “Biology and Conservation of Horseshoe Crabs”).

Dr. David Hussong and Melissa Stappen of ValSource, LLC who wrote an article called “The LAL Assay for Pyrogen Testing of Parenteral Products: Evolution & Challenges”. They answered our questions concerning the current standing of the FDA on LAL testing. They also talked about the role of the USP (an acknowledged standard-setting organization in the Code of Federal Regulations) in licensing LAL as part of the testing standard for the endotoxin assay. This makes it much more efficient for testing labs to assay products. However for alternative systems such as what we are doing with Recombinant Factor C as our iGEM project, a validation study must be performed for each drug being tested. In order to make these tests more easy to run, the three major pharmacopeias of the US, Japan, and Europe would have to figure out uniform standards for test protocols and products. Secondly, they talked about the conditions that can cause the LAL test to be faulty. These factors can range from salts, detergents, acidity, osmotic conditions, viscosity, and the nature of drug ingredients. They described how diluting your drug sample can counteract these interferences. However, the main limit to LAL testing is the reference standard endotoxin (of the USP), which is very dated. Thirdly, they said that the only way that the horseshoe crab bleeding can be replaced is if the LAL assay is precisely as effective, if not better, than the existing method at detecting pyrogens Fourthly, they explained that the mortality rate of the crabs was different depending on the method and organization releasing the statistics. There are other commercial uses for horseshoe crabs (like culinary and fish bait industries) that have no survival rate and the elimination of the biomedical industry’s need for them would greatly reduce the stress on the population. Fifthly, they stated that there were other ways to detect the endotoxin and we did not need to synthesize the coagulation part of the reaction. We could do a color-changing or turbidimetric reaction. They would need to be highly standardized. Lastly, Mr. Hussong provided some examples for how individuals can help the environment. He talked about how people can be mindful about the amount of pollution and waste they are producing. At the community level, people can join organizations that focus on environmental sustainability. Also, people who live near horseshoe crab populations can help out greatly by flipping over stranded horseshoe crabs. They often land upside down after mating season and get stranded, which can be fatal if the tide does not bring them back into the water.

Dr. NG Wai-Chuen, from the Hong Kong Agriculture, Fisheries and Conservation Department that enforces regulations on plant, pesticide, and animal controls, oversees the efficient production of fishery and agricultural produce, and safeguards ecological integrity. In his email,, Dr. Wai-Chuen discussed the current status and conservation efforts of horseshoe crabs in Hong Kong. All 3 asian horseshoe crab species (Tachypleus tridentatus, Tachypleus gigas & Carcinoscorpius rotundicaua) have been recorded in Hong Kong in the 1990s; however, only 2 species (T. tridentatus & C. rotundicauda) were recorded recently in the same location. Even though there is no blood extraction in Hong Kong, they are still threatened because of coastal development, pollution and local trawling activities. Horseshoe crabs are a conservation priority under the Environmental Impact Assessment (EIA), which ensures that ecological impacts on the species are assessed and limited. A trawl ban has also been in effect since December 31st, 2012, to help reduce the by-catch of horseshoe crabs by fisherman. Dr. Wai-Chuen also talks about how participation in public events will help increase international awareness of the importance of horseshoe crabs.

Brenda R. a drug informational specialist for the FDA Division of Drug Information, which is composed to healthcare professionals and pharmacists who provide education to the general public as well as targeted audiences such as small businesses or industries.

Education

Seattle’s Mini Maker Faire Booth

On August 19, Seattle held its annual Mini Maker Faire which showcases the work of tech creatives, scientists, and other intellectuals in the Seattle area. At the Mini Maker Faire, the community lab that our team works out of, SoundBio Lab, were one of the featured makers at the event. As the SoundBio Lab was planning on hosting an educational, outreach activity at the Faire, we also thought it would be fitting to help talk with the members of our Seattle community and discuss the topics of synthetic biology as well as more specific concepts that our project is working on addressing. We had many conversations with Seattle citizens about the importance of health care and specifically having access to sterile, safe medical equipment, protecting different animal species, and the significance of biotechnology in our daily lives.

Most memorably, the people we talked to were intrigued to learn that LAL testing is used in Seattle’s own hospitals and companies. Rim Bio, a Seattle-based company that produces single-use bioprocess containers performs LAL testing on their products to ensure that customers receive clinically sterile products[1]. Namely, Rim Bio uses the Charles River Endosafe® Test that harvests horseshoe crab blood. However, Charles River unique compared with other LAL companies as it is focused horseshoe crab conservation as stated on their website:

“Once the crabs are brought into our lab, they are carefully inspected by trained employees who determine the animal’s health and maturity. After careful collection of a measured amount of blood, the crabs are returned unharmed to their natural habitat within the same day, and their blood volume rebounds quickly. These practices have allowed Charles River to achieve an industry-leading survival rate of donor animals”[2]

We also were able to connect with a lot of parents who were visiting the booth with their kids as we talked to them about Seattle Children’s Hospital, one of the best children’s hospitals in the country. Many families in the Washington area have received life-saving treatments at Seattle Children’s and like Rim Bio, Seattle Children’s also uses LAL testing for many of their products and procedures [3]. These families were surprised to hear that some of these life saving procedures were done at the expense of horseshoe crab bleeding, and were excited to learn about our project to synthetically produce Factor C.

Gasworks Park “Learn About DNA Day”

As part of the Pacific Northwest iGEM Meetup, we held an outreach event at Gasworks Park, a popular spot for families to visit. Our goal was to educate the public about the significance of our research problem, as well as spark interest towards the growing field of synthetic biology as a whole.

Our outreach activity was primarily targeted towards young children. We created a “Code Your Name with DNA” activity, where children were taught about the structure of DNA, what DNA is used for, and some of the amazing things DNA can do in synthetic biology. This activity began with students writing out their name and matching the letters of their name to a special key. In this key, each letter of the alphabet is ‘coded’ by a set of 3 nitrogen bases, similar to the formation of the polypeptide chain in RNA translation, and the kids created a bracelet that corresponds to their name and is reminiscent of the double stranded structure of DNA.

While half of the human practices team was working directly with youth, the other half of the members spoke with parents and adults that were passing by. By connecting with adults, who have a large influence in our community, we were able to describe to them the dire situation of horseshoe crabs, and potential future shortcomings in the healthcare field due to the current LAL testing. Then, we explained our proposed solution with the synthesis of Factor C in a straightforward way that nearly anyone could understand. Through teaching others about this issue, we were also able to learn from the public as well. For example, several of the parents asked us if the expenses involved in switching to synthetic endotoxin detection will reduce its accessibility to consumers, especially in low-resource settings. Prior to this, we knew our solution had to be less expensive than extracting the blood of horseshoe crabs, but by speaking directly with those whose lives will be altered by our solution, we how critical the monetary aspect of our project was. Thus, the direction of our project was slightly shifted towards not just finding an alternate solution to current LAL tests, but also on making it accessible to all people by reducing costs.

Design

Dr. Daniel Ziegler, Director of Bacillus Genetic Stock Center at the Ohio State University, gave us guidance on whether to use b. Subtilis or b. Cereus, which plasmid to use, where to insert the fragments. He advised us to use Bacillus subtilis rather than B. cereus for safety reasons and widely available genetic tools. He also recommended using the strains 1S145 or 3NA as plasmid backbones because they are not large. Large plasmids are usually unstable in B. subtilis because their replication methods often result in long single-stranded intermediates, which can lead to deletions. Dr. Zeigler reminded us to use a gram-positive ribosome binding site. For inducible expression, he provides an example of a xylose-inducible Pxyl promoter, pAX01, that integrates into lacA gene of B. subtilis with erythromycin resistance. For the pAX01 vector, he recommends cloning into 2 of the 4 sites - NotI, SacII, SacI, or BamHI - to do directional cloning. Ultimately, by obtaining feedback from Dr. Zeigler, we were able to positively alter the design of our project.

Dr. Karen McClymont, lab specialist at Clinnovate Health, provided insight on our project as well. She pointed out that we might have better luck using insect cells, but that it might be too difficult to do in such a short amount of time. She also cautioned us that Factor C was an incredibly difficult protein to work with since it was so incredibly sensitive to LPS and contamination would be virtually unavoidable in a lab setting.

[1] http://rimbio.com/quality/endotoxin-testing/
[2] http://hsc.criver.com/#lal-endotoxins
[3] https://seattlechildrenslab.testcatalog.org/show/Acid-Lipase